Bakteerien siaalihappokatalyysitiestä lisävaloa (Thesis Rhawnie Caing-Carlsson Gothenburg University )

Väitöstyö PH.D. Thesis: Structural Insight into the Bacterial Sialic Acid Catabolic Pathway Väittelijä   Rhawnie Caing-Carlsson   http://hdl.handle.net/2077/57823

Väitöstilaisuus oli 23.11. 2018 ja opponenttina, vastaväittäjänä, toimi professori Susanna Törnroth-Horsefield.  Lundin yliopistosta. Hänen tutkimusalueensa on proteiinitutkimustieteestä.  Hän esitti yhden  työnsä diäkuvassa, ja tästä näin nimen ja  yliopiston nimen. 
http://portal.research.lu.se/portal/en/persons/susanna-horsefield(5be32e41-6f77-474c-a293-c2b7b8da63c0).html

Väitös käytiin englannin kielellä.


Osatyöt, Mitä asia koskee?
 1. Bairy, S., Gopalan, L. N., Setty, T. G., Srinivasachari, S., Manjunath, L., Kumar, J. P., Sai, G.R., Sucharita, B., Nayak, V., Ghosh, S., Sathyanarayanan, N., Caing-Carlsson, R., Wahlgren, W.Y., Friemann, R., Ramaswamy, S. (2018). Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. Microb Biotechnol, 11(2), 420-428.
VISA ARTIKEL

Summary

The process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His‐tagged Gateway vector, followed by their small‐scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large‐scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.
--
 The pathogens Haemophilus influenza, Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholera have evolved complex and efficient methods to escape immune surveillance of the host by embellishing sialic acid as its surface antigen (Almagro‐Moreno and Boyd, 2009a,b). These organisms scavenge host‐derived sialic acid by importing it into their cytoplasm by a tripartite ATP‐independent periplasmic (TRAP) transporter (Severi et al., 2005; Allen et al., 2005). It can then be used either as a carbon and nitrogen source (catabolic pathway) or incorporated as a non‐reducing terminal sugar on the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) (sialylation pathway) (Mulligan et al., 2010
..

2. Caing-Carlsson, R., Goyal, P., Sharma, A., Ghosh, S., Setty, T. G., North, R. A., Friemann, R., Ramaswamy, S. (2017). Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. Acta Crystallogr F Struct Biol Commun, 73(Pt 6), 356-362.
VISA ARTIKEL
 Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5′-triphos­phate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.


3. Caing-Carlsson, R., Goyal, P., Wahlgren, W.Y., Dunevall, E., Ramaswamy, S, Friemann, R.(2018) Expression, purification and crystallization of a sialic acid tripartite ATP-independent periplasmic (TRAP) transporter. Manuscript


4. Wahlgren, W.Y., Dunevall, E., North, R.A., Paz, A., Scalise, M., Bisignano, P., Bengtsson-Palme, J., Goyal, P., Claesson, E., Caing-Carlsson, R., Andersson, R., Beis, K., Nilsson, U.J., Farewell, A., Pochini, L., Indiveri, C., Dobson, R.C.J., Abramson, J., Ramaswamy, S., Friemann, R. (2018). Substrate-bound outward-open structure of a Na(+)-coupled sialic acid symporter reveals a new Na(+) site. Nat Commun, 9(1), 1753.

VISA ARTIKEL
Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids....To facilitate the import of scavenged sialic acids, bacteria that colonise sialylated environments deploy specific transporters, including those from the ATP-binding cassette (ABC)⁶, tripartite ATP-independent periplasmic (TRAP)^7,8, major facilitator superfamily (MFS)⁹ and sodium solute symporter (SSS)¹⁰ transporter families (reviewed by North et al.¹¹)... Despite a growing understanding of the catalytic steps involved in the cleavage of sialic acids from the host cell surface and subsequent cytoplasmic processing^2,3, little is known about the molecular determinants of import.--
..Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na⁺ gradients to drive the uptake of extracellular substrates..

.... Väitöstilaisuus edessäpäin.
Opponentin tiedot eivät ole abstraktissa painettuna, mutta väitöstilaisuudessa saa tiedon.

 Tämä työ on puhtaasti molekyylibiologian alla, mutta koskee   bakteereja, jotka voivat aihuttaa ihmisille tauteja ja ja selvittää  bakteerien siaaalihappotaloutta, ja tällaisia tietoja voi soveltaa  ihmisen immuunivastetta  kohottavalla tavalla, kun bakteerien evaasiojärjestelmiä selviää.  Muutamia  ajatuksia  väitöskirjatekstistä suomennan seuraavalla otsikolla.  Bakteerien  siaalihappotalodesta  väitöstyö.